T cell precursors in the bone marrow and thymus undergo age-related declines in developmental potential that contribute to thymic involution. The central hypothesis of this proposal is that the age-related increase in expression of p16Ink4a and Arf in these populations is a key event that contributes to the decline in thymopoiesis. The goal of this proposal is to test this hypothesis and generate `proof of concept' data that targeting their expression, particularly through the use of hormones and cytokines, can be used therapeutically to rejuvenate the involuted thymus. Initial experiments in Aim 1 will define when and in which stages of pre-thymic and intra-thymic development p16Ink4a and Arf are expressed during aging and determine how their expression affects the growth, differentiation, and survival of T cell progenitors. Aim 2 will define the relative contribution of p16Ink4a and Arf to thymic involution and determine whether down-regulating their expression can reverse that process. Agents such as growth hormone (GH), Insulin Like Growth Factor-I (IGF-I), and Keratinocyte Growth Factor (KGF) have been shown in pre-clinical studies and clinical trials to rejuvenate the involuted thymus. However, the molecular basis by which they do so is incompletely understood. Based on preliminary data, Aim 3 will test the hypothesis that they mediate their effects directly or indirectly via down-regulation of p16Ink4a and Arf expression in immature thymocytes. Taken together, the data obtained from these studies will provide a molecular basis for thymic involution and a mechanistic understanding of how various clinical interventions designed to reverse that process are acting. A reduced T cell production that accompanies thymic involution is thought to be one reason for the decline in cell mediated immunity in the elderly. If this process could be better understood, it could lead to the development of therapies designed to rejuvenate the thymus. This in turn has implications for improving vaccination efficacy and restoration of T cell production following chemotherapy or bone marrow transplantation.